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Effects of Ezrin and Heat Shock Protein 70 on Apoptosis and Proliferation of Human Osteosarcoma Cells.

ABSTRACT: OBJECTIVE:To investigate the influence of knocking down ezrin expression in combination with heat shock protein (HSP)-induced immune killing on the apoptosis and proliferation of mouse osteosarcoma cells. METHODS:The HSP70 and ezrin-shRNA DNA fragments cloned into the expression vector pGFP-V-RS and the expression vectors pGFP-V-RS-shRNA and pGFP-V-RS-shRNA-HSP70 constructed and transfected into MG63 cell line, where their status was observed by fluorescent microscopy. Expression of ezrin and HSP70 was determined by RT-PCR and western blot. Changes in cell apoptosis and proliferation were assessed by flow cytometry and MTS and changes in expression of apoptosis and cell cycle-related proteins by western blot. Specific cytotoxic T lymphocytes (CTLs) were induced by HSP70 and its lethal effect on target MG63 tumor cells analyzed by MTS assay. RESULTS:The specific vector simultaneously downregulated ezrin and upregulated HSP70. Compared with ezrin knockdown alone, simultaneous HSP70 overexpression partially recovered the promoted cellular apoptosis and proliferation suppression by induced by ezrin knockdown; however, the apoptosis rate of MG63 cells was significantly greater than that of a negative control. In addition, ezrin-shRNA and ezrin-shRNA/HSP70 promoted expression of Bax. However, expression of these agents reduces Bcl-2 and Cyclin D1. The cytotoxic effects of CTLs on target MG63 tumor cells were significantly greater in the CTL + IL-2 + HSP70 group than the CTL + IL-2 group. CONCLUSIONS:Simultaneously knocking down ezrin and overexpressing HSP70 promotes apoptosis and inhibits proliferation of osteosarcoma cells and HSP70 induces CTL, enhancing the lethal effect on tumor cells.

SUBMITTER: Yao Q

PROVIDER: S-EPMC6583739 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Effects of Ezrin and Heat Shock Protein 70 on Apoptosis and Proliferation of Human Osteosarcoma Cells.

Yao Qin Q Zhao Hui-yi HY Xie Bo-zhen BZ

Orthopaedic surgery 20150801 3

<h4>Objective</h4>To investigate the influence of knocking down ezrin expression in combination with heat shock protein (HSP)-induced immune killing on the apoptosis and proliferation of mouse osteosarcoma cells.<h4>Methods</h4>The HSP70 and ezrin-shRNA DNA fragments cloned into the expression vector pGFP-V-RS and the expression vectors pGFP-V-RS-shRNA and pGFP-V-RS-shRNA-HSP70 constructed and transfected into MG63 cell line, where their status was observed by fluorescent microscopy. Expression ...[more]

PMID: 26311104

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Project description:Recent evidence supports a role for heat-shock protein 70 (hsp70) and the 26 S proteasome in regulating apoptosis, although the precise nature of their involvement is not known. In the present study, control and Bcl-x(L)-overexpressing, interleukin-3-dependent FL5.12 cell lines were treated with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Basal proteasome activity appeared to be approximately 30% lower in bcl-x(L) cells compared with control cells using a substrate for the chymotrypsin-like activity. However, no difference in proteasome activity was detected using substrates for the trypsin-like or peptidylglutamyl peptide-hydrolysing activities. In addition, protein levels of the 20 S proteasome beta-subunit, as determined by Western blot analyses, were similar in control and bcl-x(L) cells, leading to the conclusion that proteasome activities were the same in these two cell lines. At 24 h after treatment with 500 nM MG132, apoptosis in bcl-x(L) cells (22%) was less than that observed in control cells (34%). Concomitantly, caspase activity in control cells, as assessed by N-acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartyl-7-amino-4-methylcou marin (Ac-DEVD-AMC), was twice that observed in bcl-x(L) cells. By 48 h after MG132 treatment, apoptosis and caspase activity in bcl-x(L) cells were similar to those observed in control cells at 24 h. Proteasome inhibition stimulated increases in hsp70 protein levels in control and bcl-x(L) cells by 12 h, although the maximal increases found in bcl-x(L) cells were less. Blocking this induction with hsp70 antisense oligonucleotides potentiated apoptosis after treatment with MG132. Inhibiting caspase activity with a broad-spectrum caspase inhibitor, t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone, prevented MG132-induced apoptosis. The more specific caspase-3 inhibitor, Ac-DEVD-aldehyde, afforded less protection, although both inhibitors completely inhibited Ac-DEVD-AMC cleavage. These data indicate that both hsp70 and Bcl-x(L) provide some protection against proteasome inhibitor-induced apoptosis.

Dataset Information

Effects of Ezrin and Heat Shock Protein 70 on Apoptosis and Proliferation of Human Osteosarcoma Cells.

Publications

Effects of Ezrin and Heat Shock Protein 70 on Apoptosis and Proliferation of Human Osteosarcoma Cells.

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